Sphingosine 1 phosphate receptor modulators

ABSTRACT

Compounds are provided having the structure of Formula (I): or a pharmaceutically acceptable salt, homolog, hydrate or solvate thereof, wherein R is as defined herein. Such compounds serve as modulators of the sphingosine-1-phosphate receptor, and have utility for treatment of a malcondition for which activation of this receptor is medically indicated.

BACKGROUND Technical Field

Modulators of the sphingosine-1-phosphate receptor are provided fortreatment of a malcondition for which activation of the same ismedically indicated.

Description of the Related Art

The S1P₁/EDG₁ receptor is a G-protein coupled receptor (GPCR) and is amember of the endothelial cell differentiation gene (EDG) receptorfamily. Endogenous ligands for EDG receptors include lysophospholipids,such as sphingosine-1-phosphate (SIP). Like all GPCRs, ligation of thereceptor propagates second messenger signals via activation ofG-proteins (alpha, beta and gamma). Development of small molecule S1P₁agonists and antagonists has provided insight into some physiologicalroles of the S1P₁/S1P-receptor signaling system. To this end, S1Preceptors are divided into five subtypes (i.e., S1P₁, S1P₂, S1P₃, S1P₄and S1P₅), which subtypes are expressed in a wide variety of tissues andexhibit different cell specificity. Agonism of the S1P₁ receptorperturbs lymphocyte trafficking, sequestering them in lymph nodes andother secondary lymphoid tissue. This leads to rapid and reversiblelymphopenia, and is probably due to receptor ligation on both lymphaticendothelial cells and lymphocytes themselves (Rosen et al, Immunol.Rev., 195:160-177, 2003).

BRIEF SUMMARY

In brief, modulators of the sphingosine-1-phosphate receptor areprovided for treatment of a malcondition for which activation of thesame is medically indicated.

In one embodiment, a compound is provided having the structure ofFormula (I):

or a pharmaceutically acceptable salt, homolog, hydrate or solvatethereof, wherein R is as defined below.

DETAILED DESCRIPTION OF THE DISCLOSURE

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Further, the words “comprising,” “including” and“having” are open-ended terms as used herein, and do not preclude theexistence of additional elements or components.

The present invention is directed to compounds which modulate an S1Preceptor, as well as to related products and methods for theirpreparation and use. S1P receptors are divided into five subtypes (i.e.,S1P₁, S1P₂, S1P₃, S1P₄ and S1P₅), which subtypes are expressed in a widevariety of tissues and exhibit different cell specificity. The compoundsdisclosed herein modulate one or more of these subtypes. In oneembodiment, the compounds are “S1P₁” modulators as they modulate subtype1 of a sphingosine-1-phosphate receptor. In another embodiment, thecompounds modulate subtype 1 and another subtype, such as subtype 5. Asused herein, an “S1P₁ modulator” is understood to encompass compoundsthat modulate the S1P₁ subtype alone, or modulate the S1P₁ subtype aswell as one or more other subtypes. In one embodiment, an S1P₁ modulatormodulates both the S1P₁ subtype and the S1P₅ subtype.

As used herein, a “modulator” of the S1P₁ receptor is a compound which,when administered to a subject, provides the desired integration withthe target receptor, either by way of the compound acting directly onthe receptor itself, or by way of a metabolite of the compound acting onthe receptor. Upon administration to a subject, the compounds of thisinvention modulate the S1P₁ receptor by activating on the receptor forsignal transduction. Such compounds are also referred to herein as“agonists” or “S1P₁ agonists”. Such S1P₁ agonists can be selective foraction on S1P₁. For example, a compound selective for action on S1P₁acts at a lower concentration on S1P₁ than on other subtypes of the S1Preceptor family.

Receptor agonists may be classified as either orthosteric or allosteric,and S1P₁ agonists of this invention include both classifications, eitherby way of the compound or by way of a metabolite of the compound actingon the receptor. In certain embodiments, compounds of the invention areorthostatic agonists. An orthosteric agonist binds to a site in thereceptor that significantly overlaps with the binding of the naturalligand and replicates the key interactions of the natural ligand withthe receptor. An orthosteric agonist will activate the receptor by amolecular mechanism similar to that of the natural ligand, will becompetitive for the natural ligand, and will be competitivelyantagonized by pharmacological agents that are competitive antagonistsfor the natural ligand.

In certain other embodiments, compounds of the invention are allostericagonists. An allosteric agonist binds to a site in the receptor thatmakes some significant interactions that are partly or whollynon-overlapping with the natural ligand. Allosteric agonists are trueagonists and not allosteric potentiators. Consequently, they activatereceptor signaling alone and without a requirement for a sub-maximalconcentration of the natural ligand. Allosteric agonists may beidentified when an antagonist known to be competitive for theorthosteric ligand shows non-competitive antagonism. The allostericagonist site can also be mapped by receptor mutagenesis.

In one embodiment, a compound is provided having the structure ofFormula (I):

or a pharmaceutically acceptable salt, homolog, hydrate or solvatethereof,

wherein:

R is alkyl.

As used in Formula (I), the following terms have the meanings set forthbelow.

“Alkyl” means straight chain, branched or cyclic alkyl group(cycloalkyl), saturated or unsaturated, having from 1 to about 20 carbonatoms (C₁₋₂₀ alkyl), and from 3 to 20 carbon atoms in the case ofcycloalkyl. Alkyls are typically from 1 to 12 carbons (C₁₋₁₂ alkyl) or,in some embodiments, from 1 to 8 carbon atoms (C₁₋₈ alkyl) or, in someembodiments, from 1 to 4 carbon atoms (C₁₋₄ alkyl) or, in someembodiments, from 1 to 3 carbon atoms (C₁₋₃ alkyl). Examples of straightchain alkyl groups include, but are not limited to methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups.Examples of branched alkyl groups include, but are not limited to,isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and2,2-dimethylpropyl groups.

Examples of unsaturated alkyls include alkenyl and alkynyl groups.Examples of cycloalkyl include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.In some embodiments, the cycloalkyl group has 3 to 8 ring members,whereas in other embodiments the number of ring carbon atoms range from3 to 5, 3 to 6, or 3 to 7. Cycloalkyl groups further include polycycliccycloalkyl groups such as, but not limited to, norbornyl, adamantyl,bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused ringssuch as, but not limited to, decalinyl, and the like.

In one embodiment, a compound is provided having the structure ofFormula (I), or a pharmaceutically acceptable salt, homolog, hydrate orsolvate thereof, alkyl is a straight chain or branched saturated alkylhaving from 1 to 8 carbon atoms (C₁₋₈ alkyl) or, in some embodiments,from 1 to 4 carbon atoms (C₁₋₄ alkyl) or, in some embodiments, from 1 to3 carbon atoms (C₁₋₃ alkyl). In a more specific embodiment, alkyl ismethyl, ethyl, n-propyl, n-butyl, iso-propyl, iso-butyl, sec-butyl ort-butyl.

In one embodiment, a compound is provided having the structure ofFormula (I), or a pharmaceutically acceptable salt, homolog, hydrate orsolvate thereof, wherein alkyl is cycloalkyl having from 3 to 8 ringmembers or, in some embodiments, 3 to 7, 3 to 6, or 3 to 5 ring member.In more specific embodiments, cycloalklyl is cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl.

Representative compounds of Formula (I) are listed in Table 1.

TABLE 1 Cpd No. Structure 2

3

4

5

As mentions above, compounds having the structure of Formula (I) alsoinclude pharmaceutically acceptable salts, homologs, hydrates andsolvates thereof.

A “salt” as is well known in the art includes an organic compound suchas a carboxylic acid, a sulfonic acid, or an amine, in ionic form, incombination with a counterion. For example, acids in their anionic formcan form salts with cations such as metal cations, for example sodium,potassium, and the like; with ammonium salts such as NH₄ ⁺ or thecations of various amines, including tetraalkyl ammonium salts such astetramethylammonium and alkyl ammonium salts such as tromethamine salts,or other cations such as trimethylsulfonium, and the like. A“pharmaceutically acceptable” or “pharmacologically acceptable” salt isa salt formed from an ion that has been approved for human consumptionand is generally non-toxic, such as a chloride salt or a sodium salt. A“zwitterion” is an internal salt such as can be formed in a moleculethat has at least two ionizable groups, one forming an anion and theother a cation, which serve to balance each other. For example, aminoacids such as glycine can exist in a zwitterionic form. A “zwitterion”is a salt within the meaning herein. The compounds of the presentdisclosure may take the form of salts. The term “salts” embracesaddition salts of free acids or free bases which are compounds of thedisclosure. Salts can be “pharmaceutically-acceptable salts.” The term“pharmaceutically acceptable salt” refers to salts which possesstoxicity profiles within a range that affords utility in pharmaceuticalapplications. Pharmaceutically unacceptable salts may nonethelesspossess properties such as high crystallinity, which have utility in thepractice of the present disclosure, such as for example utility inprocess of synthesis, purification or formulation of compounds of thedisclosure.

Suitable pharmaceutically acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic,sulfuric, and phosphoric acids. Appropriate organic acids may beselected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,trifluoromethanesulfonic, 2 hydroxyethanesulfonic, p toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, stearic, alginic, R hydroxybutyric,salicylic, galactaric and galacturonic acid. Examples ofpharmaceutically unacceptable acid addition salts include, for example,perchlorates and tetrafluoroborates.

Suitable pharmaceutically acceptable base addition salts of compounds ofthe disclosure include, for example, metallic salts including alkalimetal, alkaline earth metal and transition metal salts such as, forexample, calcium, magnesium, potassium, sodium and zinc salts.Pharmaceutically acceptable base addition salts also include organicsalts made from basic amines such as, for example,N,N′dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. Examples ofpharmaceutically unacceptable base addition salts include lithium saltsand cyanate salts. Although pharmaceutically unacceptable salts are notgenerally useful as medicaments, such salts may be useful, for exampleas intermediates in the synthesis of compounds, for example in theirpurification by recrystallization. All of these salts may be prepared byconventional means from the corresponding compound by reacting, forexample, the appropriate acid or base with the compound. The term“pharmaceutically acceptable salts” refers to nontoxic inorganic ororganic acid and/or base addition salts, see, for example, Gould et al.,Salt Selection for Basic Drugs (1986), Int J. Pharm., 33, 201-217,incorporated by reference herein.

Non-limiting examples of potential salts of this disclosure include butare not limited to hydrochloride, citrate, glycolate, fumarate, malate,tartrate, mesylate, esylate, cinnamate, isethionate, sulfate, phosphate,diphosphate, nitrate, hydrobromide, hydroiodide, succinate, formate,acetate, dichloroacetate, lactate, p-toluenesulfonate, pamitate,pidolate, pamoate, salicylate, 4-aminosalicylate, benzoate, 4-acetamidobenzoate, glutamate, aspartate, glycolate, adipate, alginate, ascorbate,besylate, camphorate, camphorsulfonate, camsylate, caprate, caproate,cyclamate, laurylsulfate, edisylate, gentisate, galactarate, gluceptate,gluconate, glucuronate, oxoglutarate, hippurate, lactobionate, malonate,maleate, mandalate, napsylate, napadisylate, oxalate, oleate, sebacate,stearate, succinate, thiocyanate, undecylenate, and xinafoate.

A “homolog” of a compound of the disclosure is a compound having one ormore atoms of the compound replaced by an isotope of such atom. Forexample, homologs include compounds with deuterium in place of one ormore hydrogen atoms of the compound such as compounds of the disclosurein which the methyl groups of the isopropoxy moiety of Formulas I-R andI-S are fully or partially deuterated (e.g., (D₃C)₂CHO—). Isotopicsubstitutions which may be made in the formation of homologs of thedisclosure include non-radioactive (stable) atoms such as deuterium andcarbon 13, as well as radioactive (unstable) atoms such as tritium,carbon 14, iodine 123, iodine 125, and the like.

A “hydrate” is a compound that exists in a composition with watermolecules. The composition can include water in stoichiometricquantities, such as a monohydrate or a dihydrate, or can include waterin random amounts. As the term is used herein a “hydrate” refers to asolid form, i.e., a compound in water solution, while it may behydrated, is not a hydrate as the term is used herein.

A “solvate” is a similar composition except that a solvent other thatwater replaces the water. For example, methanol or ethanol can form an“alcoholate”, which can again be stoichiometric or non-stoichiometric.As the term is used herein a “solvate” refers to a solid form, i.e., acompound in solution in a solvent, while it may be solvated, is not asolvate as the term is used herein.

The compound disclosed herein can be prepared by techniques known to oneskilled in the art, as well as by the procedures disclosed in thefollowing Examples.

EXAMPLES General Methods of Synthesis

¹H NMR (400 MHz) and ¹³C NMR (100 MHz) were obtained in solution ofdeuteriochloroform (CDCl₃), deuteriomethanol (CD₃OD) or dimethylsulfoxide—D₆ (DMSO). NMR spectra were processed using Mestrec 5.3.0 and6.0.1. ¹³C NMR peaks that are bracketed are two rotomers of the samecarbon. Mass spectra (LCMS) were obtained using an Agilent 1100/6110HPLC system equipped with a Thompson ODS-A, 100A, 5 μ (50×4.6 mm) columnusing water with 0.1% formic acid as the mobile phase A, andacetonitrile with 0.1% formic acid as the mobile phase B. The gradientwas 20-100% with mobile phase B over 2.5 min then held at 100% for 2.5mins. The flow rate was 1 mL/min. For more hydrophobic compounds, thefollowing gradient was used, denoted as Method 1: 40-95% over 0.5 min,hold at 95% for 8.5 min, then return to 40% over 2 min, with a flow rateof 1 mL/min. Final compounds were checked for purity using Method 2: 5%for 1 min, 5-95% over 9 min, then hold at 95% for 5 min, with a flowrate of 1 mL/min. Enantiomeric excess was determined by integration ofpeaks that were separated on a Chiralpak AD-H, 250×4.6 mm column, 5 pmparticle size. Flow rate of 1 mL/min and an isocratic mobile phase.Unless otherwise indicated, the chiral data provided uses this method.Alternatively, chiral separations were performed under the followingconditions, denoted as Chiral Method 1: Chiralpak AY-H, 250×4.6 mmcolumn, 5 pm particle size. Flow rate of 1 mL/min and an isocraticmobile phase. Chiral Method 2: Chiralcel OZ-3, 250×4.6, 3 μm particlesize at a flow rate of 0.75 ml/min. The pyridine, dichloromethane (DCM),tetrahydrofuran (THF), and toluene used in the procedures were fromAldrich Sure-Seal bottles kept under nitrogen (N₂). All reactions werestirred magnetically and temperatures are external reactiontemperatures. Chromatographies were carried out using a Combiflash Rfflash purification system (Teledyne Isco) equipped with Redisep(Teledyne Isco) silica gel (SiO₂) columns. Preparative HPLCpurifications were done on Varian ProStar/PrepStar system using watercontaining 0.05% trifluoroacetic acid as mobile phase A, andacetonitrile with 0.05% trifluoroacetic acid as mobile phase B. Thegradient was 10-80% with mobile phase B over 12 min, hold at 80% for 2min, and then return to 10% over 2 min with flow rate of 22 mL/min.Other methods similar to this may have been employed. Fractions werecollected using a Varian Prostar fraction collector and were evaporatedusing a Savant SpeedVac Plus vacuum pump. Microwave heating wasperformed using a Biotage Initiator microwave reactor equipped withBiotage microwave vessels. The following abbreviations are used: ethanol(EtOH), carbonyldiimidazole (CDI), isopropanol (IPA), and4-dimethylaminopyridine (DMAP).

Example 1 Synthesis of Compound No. 1

Step 1—Synthesis of 3-ethoxy-1H-indene-7-carbonitrile (Int 2)

A stirred mixture of 1-oxo-2,3-dihydro-1H-indene-4-carbonitrile (Int 1)(20.0 g, 98 wt %, 18.6 assay g, 124.8 mmol) in abs EtOH (20 mL),triethylorthoformate (80 mL, 481 mmol) and methanesulfonic acid (0.88mL, 12.5 mmol) in toluene (80 mL) was heated at 43-47° C. After 1 h, GCanalysis showed orthoformate consumed and 12.8 area % of Int 1remaining. A further charge of triethylorthoformate (20 mL, 120.2 mmol)was made and after 45 min GC analysis showed 1.5 area % Int 1. The batchwas cooled to ambient temperature and then poured into 1 M aq. K₂HPO₄(200 mL) with vigorous stirring while maintaining a quench temperature<15° C. The two-phase mixture was vigorously stirred for 10 min. Thephases were separated and the aqueous phase (pH 11) was back extractedwith toluene (100 mL). The organic phases were combined and distilled atatmospheric pressure to remove 340 mL distillate. Toluene was added (500mL) and distilled at atmospheric pressure to remove 500 mL distillate.Total distillation time 3 h, temperature range 80-120° C. At this pointthe batch was stored overnight at <5° C. Excess orthoformate was removedby chasing with ethyl acetate (100 mL) under reduced pressure untildistillation stopped. Another volume of ethyl acetate (100 mL) was addedand then concentrated under reduced pressure until distillation stopped.A third volume of ethyl acetate (100 mL) was added and then concentratedunder reduced pressure until distillation stopped, after which GCanalysis confirmed no orthoformate remaining. The crude was then stirredat 110° C. for 1 h, to convert the intermediate ketal to3-ethoxy-1H-indene-7-carbonitrile (Int 2). Upon cooling, the crude(mobile oil, 21.34 g) was assayed for Int 2 by ¹H NMR employingmesitylene as an internal standard. The oil assayed at 78.1 wt %product=16.73 assay g, 90.0 mmol=72.1% assay yield. The crude oil wasthen purified by filtration through a silica gel plug eluting with 15%EtOAc/hexane. The pure fractions were combined and utilized for the nextstep. ¹H NMR (400 MHz, d₆-DMSO) δ 7.78 (d, J=8.4, 1H), 7.63 (m, 1H),7.49 (m, 1H), 5.60 (m, 1H), 1.38 (t, J=6.8 Hz, 1H), 1.19 (t, J=6.8 Hz,1H); LRMS: calcd for C₁₂H₁₂NO⁺ [M+H]: 186.2; Found: 186.2.

Step 2—Synthesis of Int 3

An EtOAc/hexane solution (650 mL) of 3-ethoxy-1H-indene-7-carbonitrile(Int 2) is concentrated under reduced pressure to ˜17 mL and isopropylalcohol (IPA, 40 mL) was added. The solution was concentrated to ˜17 mL,and a second volume of IPA (34 mL) was added. To the stirred solutionwas added aqueous hydroxylamine (50%, 30 mL, 455 mmol). The batch wasthen warmed at 35-40° C. for 5 h, and then stirred at ambienttemperature overnight. The batch was cooled to 0° C., seeded (50 mg),and stirred for 30 min for a seed bed to develop. Water (250 mL) wasthen added dropwise over ˜ 1.5 h. The batch was stirred for 1h at 0-20°C. The product was isolated by filtration, cake-washed with water (100mL) and dried on the filter under vacuum and a nitrogen atmosphere, toafford 3-ethoxy-N-hydroxy-1H-indene-7-carboximidamide (Int 3) (20.8 g,90% yield). ¹H NMR (400 MHz, d₆-DMSO) δ 9.61 (s, 1H), 7.43 (m, 1H), 7.32(m, 2H), 5.77 (s, 1H), 5.41 (s, 1H), 4.08 (q, J=6.8 Hz, 2H), 3.45 (s,2H), 1.39 (t, J=6.8 Hz, 3H); LRMS: calcd for C₁₂H₁₅N₂O₂ ⁺ [M+H]: 219.2;Found: 219.1.

Step 3—Synthesis ofN-((3-cyano-4-isopropoxybenzoyl)oxy)-3-ethoxy-1H-indene-7-carboximidamide(Int 4)

A mixture of CDI (16.64 g, 102.6 mmol) and 3-cyano-4-isopropoxyl benzoicacid (21.06 g 102.6 mmol) in DMF (83 mL) was stirred at 20° C. for 1 h.A solution of 3-ethoxy-N-hydroxy-1H-indene-7-carboximidamide (Int 3)(20.8 g, 93.3 mmol) in DMF (40 mL) was added through an addition funnelover ˜5 min. After ˜30 min the batch became viscous and a further volumeof DMF (40 mL) was added to aid stirring. At this point HPLC assayindicated that the reaction was complete. The resulting slurry wasdiluted with water (1.5 L), cooled to 0° C., and isolated by filtration.The filter cake was washed with water (1.5 L) and the product dried onthe filter under nitrogen flow to affordN-((3-cyano-4-isopropoxybenzoyl)oxy)-3-ethoxy-1H-indene-7-carboximidamide(Int 4) as an off white solid (34.8 g, 90% yield). ¹H NMR (400 MHz,d₆-DMSO) δ 8.70 (s, 1H), 8.33 (d, J=6.8 Hz, 1H), 7.45 (m, 4H), 7.10 (m,2H), 5.49 (s, 1H), 4.94 (m, 1H), 4.10 (q, J=6.8 Hz, 2H), 3.55 (s, 2H),1.38 (m, 9H); LRMS: calcd for C₂₃H₂₄N₃O₄ ⁺ [M+H]: 406.4; Found: 406.2.

Step 4—Synthesis of5-(3-(3-ethoxy-1H-inden-7-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile(Int 5)

N-((3-Cyano-4-isopropoxybenzoyl)oxy)-3-ethoxy-1H-indene-7-carboximidamide(Int 4) (34.8 g, 83.97 mmol) was suspended in toluene (590 mL) andheated to reflux with a Dean-Stark apparatus for 18 h. ˜2 mL werecollected (theory 1.5 mL). The batch was cooled to ambient temperature,filtered through Celite, and concentrated under vacuum. The crude solid5-(3-(3-ethoxy-1H-inden-7-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile(Int 5) (30 g, 90% yield) is taken as is to the next step. LRMS: calcdfor C₂₃H₂₂N₃O₃ ⁺ [M+H]: 388.4; Found: 388.3.

Step 5—Synthesis2-isopropoxy-5-(3-(1-oxo-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)benzonitrile(Cpd. No. 1)

Int 5 (30 g, 75.57 mmol) is suspended in 4:1 IPA/H₂O (300 mL). CatalyticH₂SO₄ (0.1 mL, 0.19 mmol) is added, and the resulting mixture is heatedto reflux for 12 h. The slurry is cooled to ambient temperature andstirred for 1 h. The product is isolated by filtration and washed with4:1 IPA/H₂O (100 mL). After drying on the filter for 1 h under vacuum,the wet cake is charged back to the reactor and suspended in EtOAc (300mL). The mixture is heated to reflux for 3 h, then cooled to ambienttemperature and stirred for 1 h. The slurry is filtered, washed withEtOAc (100 mL), and dried on the filter under nitrogen to afford2-isopropoxy-5-(3-(1-oxo-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)benzonitrile(Cpd. No. 1) (22 g, 80% yield) as an off-white solid. ¹H NMR (400 MHz,d₆-DMSO) δ 8.55 (d, J=2.0 Hz, 1H), 8.44 (m, 2H), 7.88 (d, J=7.6 Hz, 1H),7.69 (t, J=7.6 Hz, 1H), 7.57 (d, J=9.2 Hz, 1H), 4.99 (h, J=12.4 Hz, 1H),3.46 (dd, J₁=5.6, J₂=11.2 Hz, 2H), 2.76 (dd, J₁=5.6, J₂=11.2 Hz, 2H),1.45 (d, J=12.4 Hz, 6H); ¹³C NMR (100 MHz, d₆-DMSO) δ 205.9, 173.4,167.4, 162.6, 154.2, 138.1, 134.7, 134.2, 133.9, 128.2, 125.9, 124.5,115.8, 115.3, 114.9, 102.5, 72.6, 35.9, 27.3, 21.5; LRMS: calcd forC₂₁H₁₈N₃O₃ ⁺ [M+H]: 360.1; Found: 360.2; C,H,N Analysis: Found: % C:70.25, % H: 4.69; % N: 11.71; Theory: % C: 70.18; % H: 4.77; % N: 11.69.

Example 2 Synthesis of Compound 2(5-(3-(1-hydroxy-1-methyl-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile)

2-isopropoxy-5-(3-(1-oxo-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)benzonitrile(100 mg, 0.28 mmol) in DCM (4 ml), then added Ether (4 ml), then asolution of methylmagnesium bromide ((0.158 ml, 0.4 mmol, 3M in Ether)was added to above solution at 20° C. The mixture was stirred at rt for20 min, 25% conversion observed; added 0.15 ml more of methylmagnesiumbromide (3M in Ether) and stirred for another 30 min. Then the reactionmixture was poured into ice-water. 2M HCl aqueous (5 ml) was added tothe solution to pH-1; then extracted with EtOAc (20 ml). The organiclayer was washed with brine, dried, concentrated, then purified by ISCOto provide the desired product:5-(3-(1-hydroxy-1-methyl-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxy-benzonitrile(15 mg, 0.04 mmol, 15%). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.48 (d,J=8 Hz, 6H), 1.63 (s, 3H), 2.35 (m, 2H), 3.25 (m, 1H), 3.45 (m, 1H),4.81 (m, 1H), 7.12 (d, J=8 Hz, 1H), 7.45 (t, J=4 Hz, 1H), 7.50 (d, J=8Hz, 1H), 8.23 (d, J=8 Hz, 1H), 8.35 (d, J=8 Hz, 1H), 8.46 (s, 1H); ESIMSfound for C₂₂H₂₁N₃O₃: m/z 376.0 (M+1).

Example 3 Synthesis of Compound 3(5-(3-(1-ethyl-1-hydroxy-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile)

5-(3-(1-ethyl-1-hydroxy-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrilewas prepared in accordance with the procedures described in Example 2,except methylmagnesium bromide was replaced by ethylmagnesium bromide in23% yield. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.0 (m, 3H), 1.48 (d,J=8 Hz, 6H), 1.84 (m, 1H), 2.01 (m, 1H), 2.15 (m, 1H), 2.45 (m, 1H),3.25 (m, 1H), 3.45 (m, 1H), 4.81 (m, 1H), 7.12 (d, J=8 Hz, 1H), 7.45 (t,J=4 Hz, 1H), 7.50 (d, J=8 Hz, 1H), 8.23 (d, J=8 Hz, 1H), 8.35 (d, J=8Hz, 1H), 8.46 (s, 1H); ESIMS found for C₂₃H₂₃N₃O₃: m/z 390.0 (M+1).

Example 4 Synthesis of Compound 4(5-(3-(1-hydroxy-1-isopropyl-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile)

5-(3-(1-hydroxy-1-isopropyl-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile(N39-034) was prepared in accordance with the procedures described inExample 2, except methylmagnesium bromide was replaced byisopropylmagnesium bromide in 20% yield. ¹H NMR (400 MHz, CHLOROFORM-d)δ ppm 0.80 (d, J=8 Hz, 3H), 1.0 (d, J=8 Hz, 3H), 1.48 (d, J=8 Hz, 6H),2.05 (m, 1H), 2.25 (m, 1H), 2.49 (m, 1H), 3.25 (m, 1H), 3.45 (m, 1H),4.81 (m, 1H), 7.12 (d, J=8 Hz, 1H), 7.45 (t, J=4 Hz, 1H), 7.50 (d, J=8Hz, 1H), 8.23 (d, J=8 Hz, 1H), 8.35 (d, J=8 Hz, 1H), 8.46 (s, 1H); ESIMSfound for C₂₄H₂₅N₃O₃: m/z 404.7 (M+1).

Example 5 Synthesis of Compound 5(5-(3-(1-cyclopropyl-1-hydroxy-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile)

2-isopropoxy-5-(3-(1-oxo-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)benzonitrile(200 mg, 0.56 mmol) in DCM (4 ml), then added Ether (8 ml), then asolution of cyclopropylmagnesium bromide (0.189 ml, 0.94 mmol, 0.5 M inTHF) was added to above solution at 20° C. The mixture was stirred at rtfor 20 min, 25% conversion observed. Then the reaction mixture waspoured into ice-water. 2M HCl aqueous (5 ml) was added to the solutionto pH-1; then extracted with EtOAc (20 ml). The organic layer was washedwith brine, dried, and concentrated. The crude material was treated withexcess of NaBH₄ to reduce the product to the alcohol, and then purifiedby ISCO to provide the desired product:5-(3-(1-cyclopropyl-1-hydroxy-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile(35 mg, 0.087 mmol, 16%). ¹HNMR (400 MHz, CHLOROFORM-d) δ ppm 0.50 (m,4H), 1.40 (m, 1H), 1.48 (d, J=8 Hz, 6H), 2.15 (m, 1H), 2.35 (m, 1H),3.25 (m, 1H), 3.45 (m, 1H), 4.81 (m, 1H), 7.12 (d, J=8 Hz, 1H), 7.45 (t,J=4 Hz, 1H), 7.50 (d, J=8 Hz, 1H), 8.23 (d, J=8 Hz, 1H), 8.35 (d, J=8Hz, 1H), 8.46 (s, 1H); ESIMS found for _(C24H23N3O3): m/z 384.1 (M-18).

Example 6 In Vitro Biological Assays GTPγS Binding Assay

Binding assays for [35S]-GTPTS were performed in 96-well non-bindingsurface plates with a final volume of 200 μL. The test compounds wereserial diluted in DMSO and added to assay plates using a Tecan D300Edigital printer with a total volume of 0.4 μL. The controlsphingosine-1-phosphate (S1P) was prepared separately by preparing a 400μM stock solution from a 100 nmol pellet of S1P in 10 mM Na₂CO₃ with 2%O-cyclodextrin. The serial dilution of S1P was done using complete assaybuffer (20 mM HEPES, 10 mM MgCl₂, 100 mM NaCl, 1 mM EDTA, 0.1% fattyacid free bovine serum albumin [BSA], and 30 μg/mL saponin, pH7.4) andtransferred to wells already containing 0.4 μL DMSO. All the wells werethen loaded to a total volume of 40 μL of complete assay buffer, exceptthe non-specific binding (NSB) wells. For NSB wells, 40 L/well of 50 μMGTPTS (Sigma Aldrich, cat # G8634, St. Louis, Mo.) was added to wellscontaining 0.4 μL of DMSO. The assay was started by the addition of 120μL/well of CHO-S1P receptor membrane solution containing 40 μg/mL ofmembrane protein, 16.67 μM guanosine diphosphate (GDP; Sigma Aldrich,cat # G7127, St. Louis, Mo.), and 2.5 mg/mL of WGA PVT SPA beads incomplete buffer. Assay plates were then sealed and incubated at roomtemperature with gentle agitation for 30 minutes. Next, 40 μL/well of 1nM of [³⁵S]-GTPTS (PerkinElmer, cat # NEG030X250UC, Waltham, Mass.) inbasic assay buffer (20 mM HEPES, 10 mM MgCl₂, 100 mM NaCl, and 1 mMEDTA, pH7.4) was added to the assay plates to yield a finalconcentration of 200 μM and the plates were further incubated for 40minutes at room temperature with gentle agitation. The assay wasterminated by centrifugation of the plates at 1000 rpm for 3 minutesusing an Eppendorf 5810R centrifuge (Eppendorf, Hamburg, Germany) and Gprotein bound radioactivity was quantitated using a MicroBeta2microplate scintillation counter (PerkinElmer, Waltham, Mass.).

The data for representative compounds assayed by the above technique arepresented in Table 2.

TABLE 2 S₁P₁ S₁P₅ Cpd. No. EC₅₀ (uM) % Efficacy EC₅₀ (uM) % Efficacy 30.014 97 0.147 80 4 0.007 97 0.077 74

Example 7 In Vivo Biological Assays Determination of Absolute OralBioavailability in Rats.

Pharmacokinetic studies are conducted in non-fasted male Sprague-Dawelyrats (Simonsen Laboratories or Harlan Laboratories). Rats are housed inan ALAAC accredited facility and the research approved by the facilitiesInstitutional Animal Care and Use Committee (IACUC). The animals areacclimated to the laboratory for at least 48 h prior to initiation ofexperiments.

Compounds are formulated in 5% DMSO/5% Tween20 and 90% purified water(intravenous infusion) or 5% DMSO/5% Tween20 and 90% 0.1N HCL (oralgavage). The concentration of the dosing solutions is verified byHPLC-UV. For intravenous dosing, compounds were administered by aninfusion pump into the jugular vein over one minute to manuallyrestrained animals (n=4 rats/compound). Oral dosing is by gavage using astandard stainless steel gavage needle (n=2-4 rats/compound). For bothroutes of administration, blood is collected at eight time-points afterdosing with the final sample drawn 24 h post dose. Aliquots of the bloodsamples are transferred to polypropylene 96-well plate and frozen at−20° C. until analysis.

After thawing the blood samples at room temperature, 5 μL of DMSO isadded to each well. Proteins are precipitated by adding 150 μLacetonitrile containing 200 μnM internal standard(4-hydroxy-3-(alpha-iminobenzyl)-1-methyl-6-phenylpyrindin-2-(1H)-one)and 0.1% formic acid. Plates are mixed for 1 min on a plate shaker tofacilitate protein precipitation and then centrifuged at 3,000 rpm for10 min to pellet protein. The supernatant is transferred to a cleanplate and centrifuged at 3,000 rpm for 10 min to pellet any remainingsolid material prior to LC/MS/MS analysis. Calibration curve standardsare prepared by spiking 5 μL compound stock in DMSO into freshlycollected EDTA rat blood. An eight point standard curve spanning a rangeof 5 nM to 10,000 nM is included with each bio-analytical run. Thestandards are processed identically to the rat pharmacokinetic samples.

Concentrations in the rat pharmacokinetic samples are determined using astandardized HPLC-LC/MS/MS method relative to the eight point standardcurve. The system consists of a Leap CTC Pal injector, Agilent 1200 HPLCwith binary pump coupled with an Applied Biosystems 3200 QTrap.Compounds are chromatographed on a Phenomenex Synergy Fusion RP 20×2 mm2 um Mercury Cartridge with Security Guard. A gradient method is usedwith mobile phase A consisting of 0.1% formic acid in water and mobilephase B consisting of 0.1% formic acid in acetonitrile at flow ratesvarying from 0.7 to 0.8 mL/min. Ions are generated in positiveionization mode using an electrospray ionization (ESI) interface.Multiple reaction monitoring (MRM) methods are developed specific toeach compound. The heated nebulizer is set at 325° C. with a nebulizercurrent of 4.8 μA. Collision energies are used to generate daughter ionsranged between 29 and 39 V. Peak area ratios are obtained from MRM ofthe mass transitions specific for each compound used for quantification.The limit of quantification of the method is typically 5 nM. Data arecollected and analyzed using Analyst software version 1.4.2.

Blood concentration versus time data are analyzed usingnon-compartmental methods (WinNonlin version 5.2; model 200 for oraldosing and model 202 for intravenous infusion). Absolute oralbioavailability (%) is calculated using the following expression: (OralAUC×IV Dose)/(IV AUC×Oral Dose)×100.

Lymphopenia

In mice: Female C57BL6 mice (Simonsen Laboratories, Gilroy CA) arehoused in an ALAAC accredited facility and the research was approved bythe facilities Institutional Animal Care and Use Committee (IACUC). Theanimals are acclimated to the laboratory for at least 5 days prior toinitiation of experiments. Mice (n=3/compound/time-point) are dosed byoral gavage with 1-30 mg/kg compound formulated in a vehicle consistingof 5% DMSO/5% Tween 20 and 90% 0.1N HCl. Control mice are dosed PO withthe vehicle. Terminal whole blood samples are collected from isofluraneanesthetized mice by cardiac puncture into EDTA. Whole blood isincubated with rat anti-mouse CD16/CD32 (Mouse BD Fc Block, #553141),PE-Rat anti-mouse CD45R/B220 (BD #553089), APC-Cy7-Rat anti-mouse CD8a(BD #557654), and Alexa Fluor647-Rat anti-mouse CD4 (BD #557681) for 30min on ice. Red blood cells are lysed using BD Pharm Lyse Lysing buffer(#555899) and white blood cells were analyzed by FACS. Lymphopenia isexpressed as the % of white blood cells that were CD4 or CD8 positive Tcells. The overall lymphopenia response over 24 h is estimated bycalculating the area under the effect curve (AUEC) using the lineartrapezoidal rule.

In rats: Male rats (Simonsen Laboratories, Gilroy CA) are housed in anALAAC accredited facility and the research was approved by thefacilities Institutional Animal Care and Use Committee (IACUC). Theanimals are acclimated to the laboratory for at least 5 days prior toinitiation of experiments. Rats (n=3/compound/time-point) are dosed byoral gavage with 1-30 mg/kg compound formulated in a vehicle consistingof 5% DMSO/5% Tween 20 and 90% 0.1N HCL. Control rats are dosed PO withthe vehicle. Whole blood is collected from isoflurane anesthetized ratsvia the retro-orbital sinus and terminal samples were collected bycardiac puncture into EDTA. Whole blood is incubated with mouse anti-ratCD32 (BD #550271), PE-mouse anti-rat CD45R/B220 (BD #554881),PECy5-mouse anti-rat CD4 (BD #554839), and APC-mouse anti-rat CD8a(eBioscience #17-0084) for 30 minutes on ice. Red blood cells are lysedusing BD Pharm Lyse Lysing buffer (#555899) and white blood cells areanalyzed with a BD FACSArray. Lymphopenia is expressed as the % of whiteblood cells that were CD4 or CD8 positive T cells. The overalllymphopenia response over 24 h is estimated by calculating the areaunder the effect curve (AUEC) using the linear trapezoidal rule.

Lymphopenia

In mice: Female C57BL6 mice (Simonsen Laboratories, Gilroy CA) arehoused in an ALAAC accredited facility and the research was approved bythe facilities Institutional Animal Care and Use Committee (IACUC). Theanimals are acclimated to the laboratory for at least 5 days prior toinitiation of experiments. Mice (n=3/compound/time-point) are dosed byoral gavage with 1 mg/kg compound formulated in a vehicle consisting of5% DMSO/5% Tween 20 and 90% 0.1N HCl. Control mice are dosed PO with thevehicle. Terminal whole blood samples are collected from isofluraneanesthetized mice by cardiac puncture into EDTA. Whole blood isincubated with rat anti-mouse CD16/CD32 (Mouse BD Fc Block, #553141),PE-Rat anti-mouse CD45R/B220 (BD #553089), APC-Cy7-Rat anti-mouse CD8a(BD #557654), and Alexa Fluor647-Rat anti-mouse CD4 (BD #557681) for 30min on ice. Red blood cells are lysed using BD Pharm Lyse Lysing buffer(#555899) and white blood cells were analyzed by FACS. Lymphopenia isexpressed as the % of white blood cells that were CD4 or CD8 positive Tcells. The overall lymphopenia response over 24 h is estimated bycalculating the area under the effect curve (AUEC) using the lineartrapezoidal rule.

In rats: Female rats (Simonsen Laboratories, Gilroy CA) are housed in anALAAC accredited facility and the research was approved by thefacilities Institutional Animal Care and Use Committee (IACUC). Theanimals are acclimated to the laboratory for at least 5 days prior toinitiation of experiments. Rats (n=3/compound/time-point) are dosed byoral gavage with 1 mg/kg compound formulated in a vehicle consisting of5% DMSO/5% Tween 20 and 90% 0.1N HCL. Control rats are dosed PO with thevehicle. Whole blood is collected from isoflurane anesthetized rats viathe retro-orbital sinus and terminal samples were collected by cardiacpuncture into EDTA. Whole blood is incubated with mouse anti-rat CD32(BD #550271), PE-mouse anti-rat CD45R/B220 (BD #554881), PECy5-mouseanti-rat CD4 (BD #554839), and APC-mouse anti-rat CD8a (eBioscience#17-0084) for 30 minutes on ice. Red blood cells are lysed using BDPharm Lyse Lysing buffer (#555899) and white blood cells are analyzedwith a BD FACSArray. Lymphopenia is expressed as the % of white bloodcells that were CD4 or CD8 positive T cells. The overall lymphopeniaresponse over 24 h is estimated by calculating the area under the effectcurve (AUEC) using the linear trapezoidal rule.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments. These and other changes can be made to the embodiments inlight of the above-detailed description. In general, in the followingclaims, the terms used should not be construed to limit the claims tothe specific embodiments disclosed in the specification and the claims,but should be construed to include all possible embodiments along withthe full scope of equivalents to which such claims are entitled. U.S.Provisional Application 63/001,085, filed Mar. 27, 2020 and U.S.Provisional Application 63/018,333, filed Apr. 30, 2020 are incorporatedherein by reference, in their entirety.

We claim:
 1. A compound having the structure of Formula (I):

or a pharmaceutically acceptable salt, homolog, hydrate or solvate thereof, wherein R is alkyl.
 2. The compound of claim 1 wherein alkyl is a straight chain or branched saturated alkyl having from 1 to 8 carbon atoms.
 3. The compound of claim 2 wherein alkyl is a straight chain or branched saturated alkyl having from 1 to 4 carbon atoms.
 4. The compound of claim 3 wherein alkyl is methyl, ethyl or isopropyl.
 5. The compound of claim 1 wherein R is cycloalklyl having from 3 to 8 ring members.
 6. The compound of claim 5 wherein cycloalklyl has from 3 to 6 ring members.
 7. The compound of claim 6 wherein R is cycloalklyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
 8. The compound of claim 1 wherein the compound has one of the following structures, or a pharmaceutically acceptable salt, homolog, hydrate or solvate thereof: 